This invention relates generally to bacteria used in the fermentation of milk-containing products. More particularly, it relates to novel bacteria and bacterial compositions and methods which can be used to impart very high viscosity to fermented milk products.
By way of further background, for many years, fermented or "cultured" milk products have formed a substantial component of the human diet. For example, many cultured milk products have long been popular in North America, including for example buttermilk, sour cream, cottage cheese, dressings, pudding, yogurt and many others. Additional cultured milk products have long been popular in other areas. For example, while not well known in North America, sour milk products such as taette and long milk are popular in Sweden, and a firm, viscous cultured product known as "viili" is produced in notable quantities in Finland.
Producing these and other cultured dairy products depends on fermentation by lactic acid bacteria. As cultures of these bacteria grow in the milk product, they can impart certain characteristics such as acidity, flavor, and texture. Commonly used lactic bacteria for these purposes include strains from the genus Lactococcus, Lactobacillus, Streptococcus, Pediococcus and Leuconostoc. As will be appreciated, successful growth of these bacteria, and thus the successful production of quality endproduct, is a delicate process which can be affected by many physical and biological factors.
For instance, lactic acid bacteria are attacked by bacteriophage, which can lead to partial or complete loss of fermentations. See, for instance, Saxelin et al., "Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk `Viili`", Applied and Environmental Microbiology, October, 1986, pp. 771-777; and, U.S. Pat. No. 4,883,756 to Klaenhammer et al. Additionally, many of the desired phenotypes are encoded on extrachromosomal DNA, e.g. plasmid DNA, which results in frequent loss of desired phenotypes in culture transfers and other manipulations. Further, even assuming complete failure of fermentation can be avoided, it is often difficult to control the level of expression of a flavor, acidity, texture, etc. phenotype in a milk product, even though achieving this control can be crucial to success of endproduct. One method which has been used to control levels of expression is to combine the flavor, texture, or acid-producing bacteria with other bacteria which do not have the phenotype of interest. In this manner, the growth of the phenotypic bacteria can be inhibited by competition from the other bacteria. However, in many cases the differing types of bacteria included do not reproduce at the same rate and/or having differing phage sensitivities, and the ratio of the two types of bacteria, and thus the level of expression of the phenotype of interest, is not reliably controlled during culturing of the product.
As an example of one phenotype, the development of viscosity in cultured milk products has been of notable interest in the dairy industry. This viscosity is developed by growth of "viscous" (also commonly referred to as "slime-producing", "ropy" or "mucoid") strains of lactic bacteria. Viscous bacteria, like those responsible for other desired phenotypes, are subject to these same concerns of phage sensitivity, loss of phenotype, and control of expression in products. For example, there presently exist two primary approaches to producing viscous dairy products. One uses a viscous L. cremoris and a non-viscous lactococcal strain combined in the same culture. A second approach uses separate cultures, one containing viscous L. cremoris strains and another containing non-viscous lactococcal strains. Currently, both approaches fail to meet commercial demands for very high viscosity milk fermentations which must endure severe agitation during processing, maintain a minimal viscosity over long periods of storage, or meet higher viscosity criteria for cultured milk products or dairy based food additives.
In fermentations inoculated with a single culture containing both viscous and non-viscous lactococci, practical limits exist in the degree of viscosity that can be achieved. These limitations are a result of using lactococcal strains which inherently grow relatively slowly (L. cremoris) and/or carry plasmids which produce limited amounts of viscosity per cell. In this approach, the proportion of viscous strains must be restricted to a level where undesirable organoleptic properties are prevented. To increase endproduct viscosity, a culture manufacturer could attempt to create a culture with a higher proportion of viscous strains. However, such high proportions of viscous strains would offset the balance with non-viscous strains (strain domination), and possibly result in decreased fermentation due to bacteriophage attack. Attempts to leave cultures unchanged but alter the process to increase or maintain product viscosity have met with resistance from the industry in favor of conserving standard operating procedures and product formulations. Therefore, it has become imperative to develop new bacteria and methods which improve upon present industry standards, to meet the demand for reliable, controllable fermentations.
In general, the majority of lactic bacteria known to produce high viscosity are Lactococcus cremoris (previously Streptococcus cremoris) strains. These strains have been most often used in the production of the thick, doughy Swedish and Finnish sour milk products such as taette, long milk and viili. These viscous strains have not enjoyed widespread use in the dairy industry because it is difficult to reliably control the level of the viscous expression, and because they are particularly sensitive to loss of the viscous phenotype. See, D. Macura and P. M. Townsley, "Scandinavian Ropy Milk--Identification and Characterization of Endogenous Ropy Lactic Streptococci and Their Extracellular Excretion", J. Dairy Sci. 67: 735-744 (1984). Further, these viscous L. cremoris strains typically provide optimum viscosity when grown at a temperature of about 21.degree. C., whereas producers of numerous of the known cultured milk products have optimized fermentation temperatures at about 23.degree. to 25.degree. C. Moreover, L. cremoris strains have also have been noted for their phage sensitivity.
Recognizing the value of the viscous phenotype but at the same time the limitations of the L. cremoris strains having it, investigators have attempted to discern where the viscous phenotype is encoded in various L. cremoris strains, and to develop methods to impart this quality to other types of bacteria having more desirable and varied biological properties. In so doing, the commercial impact of the research is limited by certain standards in the dairy industry. As an example, while natural (i.e. conjugal) transfer of genetic information is acceptable for constructing new strains for food production in the U.S., other non-natural types of transfer, such as transformation, are not. In the face of these and other technical and commercial constraints, the efforts to expand the number of available viscous strains to satisfy the need for improved fermentation systems have met with limited success.
In this vein, during the mid 1980's, E. R. Vedamuthu and J. M. Neville demonstrated that the ability to produce mucoidness (Muc.sup.+) in milk cultures in strain Streptococcus cremoris MS is encoded on an 18.5M dalton plasmid which was designated as pSRQ2202. "Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS", Applied and Environmental Microbiology, April 1986, pp. 677-682. The authors reported successfully achieving conjugal transfer of pSRQ2202 from S. cremoris MS to a nonmucoid S. lactis recipient ML-3/2.201, and subsequent transfer of pSRQ2202 from the resultant S. lactis mucoid strain to malty S. lactis 4/4.2 and to S. lactis subsp. diacetylactis SLA3.25. While the accepted mode of conjugal transfer was achieved in this work, the applicant, through independent study, has determined that pSRQ2202 confers only moderate viscosity and thus leaves much room for improvement.
Report of this work by Vedamuthu and Neville was soon followed by that of A. von Wright and A. Tynkkynen, "Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype", Applied and Environmental Microbiology, June 1987, pp. 1385-1386. Von Wright and Tynkkynen reported obtaining lactose-metabolizing mucoid (Lac.sup.+ Muc.sup.+) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 by protoplast transformation with total plasmid DNA from Muc.sup.+ S. lactis subsp. cremoris ARH87. The authors concluded that the Muc.sup.+ function is encoded in a plasmid designated pVS5 from Muc.sup.+ S. lactis subsp. cremoris ARH87. Based on a restriction pattern obtained with restriction endonuclease BglII, the authors calculated the size of pVS5 to be 30 MDa, clearly larger than that of pSRQ2202 (18.5 MDa) reported by Vedamuthu and Neville in their work. Unfortunately, the type of genetic transfer (i.e. transformation) achieved by von Wright et al. is generally not acceptable to U.S. and other food producers, and thus their work is of limited commercial interest. Furthermore, these authors failed to demonstrate production of a high frequency conjugal donor of the viscous phenotype, which is essential to the development of successful commercial dairy ventures.
In 1988, work to expand the available viscous strains continued and another study of several ropy Swedish and Finnish S. cremoris strains was published. H. Neve et al., "Plasmid-encoded functions of ropy lactic acid streptococcal strains from Scandinavian fermented milk", Biochimie (1988) 70: 437-442. Neve et al. reported strong indications that the Rop.sup.+ phenotype in the Swedish strains was encoded on a 17 MDa plasmid and in the Finnish strains on a 30 MDa plasmid. Through a series of mating experiments, Neve et al. attempted to obtain conjugal transfer of these 17 and 30 MDa plasmids to S. lactis subsp. diacetylactis Bu2-59 (Rop.sup.- Lac.sup.-). However, the authors reported that no transfer of the Rop.sup.+ phenotype could be observed from any of the mating experiments in their investigation.
In light of the foregoing discussion, it is evident that there are continuing needs for improvement in the field of cultured milk products. The number of available viscous strains remains limited despite efforts to expand it. The viscous L. cremoris strains have not achieved widespread use due to undesirable traits they exhibit. Attempts to transfer their viscous phenotype to other types of bacteria have met with only limited success. While successful conjugal transfer of the 18.5 MDa plasmid pSRQ2202 to a L. lactis strain has been achieved, this plasmid encodes for only moderate viscosity, and to applicant's knowledge no interspecies conjugal transfer of any other viscous plasmid from a L. cremoris strain has been reported. In fact, Neville et al. attempted just that but were unsuccessful. The work of von Wright et al. in which a viscous plasmid was transformed from a L. lactis subsp. cremoris to a L. lactis subsp. lactis is of general scientific interest; however, this mode of genetic transfer is not considered proper for food products in the U.S., Europe and possibly other countries. Further, von Wright et al. did not demonstrate obtaining a high frequency conjugal donor of a high viscous phenotype. Such a donor is essential in achieving food grade strategies for strain development necessary for significant commercial applications. It is in light of this extensive background that the applicants entered their study, and have now succeeded in developing novel bacteria, bacterial compositions, and methods to address these and other needs in the industry.